This invention relates generally to a process of inhibiting the binding of xcex14xcex21 integrin to proteins such as VCAM-1 or fibronectin. The invention also relates to synthetic cyclic peptides that inhibit that binding.
Vascular cell adhesion molecule-1 (VCAM-1) is a protein that is found on the surface of endothelial cells that line the interior wall of capillaries. VCAM-1 recognizes and binds to the integrin xcex14xcex21 (or VLA-4 for very late antigen-4), a beterodimeric protein present on the surface of certain white blood cells. Binding of xcex14xcex21 to VCAM-1 allows white blood cells to adhere to the capillary wall in areas where the tissue surrounding the capillary has been infected or damaged.
When a tissue has been invaded by a microorganism or has been damaged, white blood cells, also called leukocytes, play a major role in the inflammatory response. One of the most important aspects of the inflammatory response involves the cell adhesion event. Generally, white blood cells are found circulating through the bloodstream. However, when a tissue is infected or becomes damaged, the white blood cells recognize the invaded or damaged tissue, bind to the wall of the capillary near the affected tissue and diffuse through the capillary into the affected tissue. VCAM-1 helps certain types of white blood cells recognize the affected sites, bind to the capillary wall, and migrate into the affected tissue.
The are are three main types of white blood cells: granulocytes, monocytes and lymphocytes. VCAM-1 binds to xcex14xcex21 expressed on the surface of monocytes, lymphocytes and two subclasses of granulocytes-eosinophils and basophils.
Monocytes, after leaving the bloodstream through the wall of a capillary, mature into macrophages that phagocytose and digest invading microorganisms, foreign bodies and senescent cells. Lymphocytes produce antibodies and kill infected cells. Eosinophils and basophils secrete mediators of various inflammatory reactions.
Following infection or damage of tissue surrounding a capillary, the endothelial cells that line the capillary express a series of adhesion molecules, including VCAM-1, that are critical for binding white blood cells that are necessary for fighting infection. Prior to binding to VCAM-1, the white blood cells initially bind to another set of adhesion molecules to slow their flow and allow the cells to xe2x80x9crollxe2x80x9d along the activated endothelium. Monocytes, lymphocytes, basophils and eosinophils are then able to firmly bind to VCAM-1 on the endothelial cells via the xcex14xcex21 integrin. There is evidence that this interaction is also involved in transmigration of these white blood cells into the damaged tissue.
Although white blood cell migration to the site of injury helps fight infection and destroy foreign material, in many instances this migration can become uncontrolled, with white blood cells flooding to the scene, causing widespread tissue damage. Compounds capable of blocking this process, therefore, would be beneficial as therapeutic agents. Thus, it would be useful to develop inhibitors that would prevent the binding of white blood cells to VCAM-1.
For example, some of the diseases that might be treated by the inhibition of xcex14xcex21 binding include, but are not limited to, atherosclerosis, rheumatoid arthritis, asthma, allergy, multiple sclerosis and type I diabetes. In addition to being found on some white blood cells, xcex14xcex21 is found on various cancer cells, including leukemia, melanoma, lymphoma and sarcoma cells. It has been suggested that cell adhesion involving xcex14xcex21 may be involved in the metastasis of certain cancers. Inhibitors of xcex14xcex21 binding may, therefore, also be useful in the treatment of some forms of cancer.
In one aspect, the present invention provides an isolated and purified cyclic peptide of from 4 to about 13 amino acid residues having (a) an N-terminal amine group, acetyl group (Ac) or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight (MPEGX000) linked through an amide bond to the N-terminal residue; and (b) a C-terminal carboxylic acid group or amide group; the peptide comprising the amino acid residue sequence Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa1 is any L- or D-xcex1-amino acid residue and Xaa2 and Xaa3 are any hydrophobic, L-xcex1-amino acid residue with the proviso that when Xaa1 is Lys or Arg, Xaa2 cannot be Gly or Cys. In a preferred embodiment, Xaa1 is Phe, Trp or Ile; Xaa2 is Leu, Ile, Val, Lys or Met and Xaa3 is Val, Tyr, Leu, Trp, or Phe. More preferably Xaa1 is Trp and Xaa2 is Leu and Xaa3 is Val.
In one embodiment, a cyclic peptide of the present invention is cyclized via formation of a lactam. Such a peptide comprises the amino acid residue sequence Xaa4-Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:1), where Xaa1 is any L- or D-xcex1-amino acid residue, Xaa2 is any hydrophobic, L-xcex1-amino acid residue with the proviso that when Xaa1 is Lys or Arg, Xaa2 cannot be Gly or Cys; Xaa3 is any hydrophobic, L-xcex1-amino acid residue and Xaa4 is any D- or L-xcex1-amino acid. Preferably, Xaa1 is Phe or Trp, Xaa2 is Leu, Xaa3 is Val and Xaa4 is Glu. More preferably, such a peptide has the amino acid residue sequence of SEQ ID NO:2-8.
In another embodiment, a cyclic peptide of this invention further comprises a cysteine or modified cysteine residue and a xe2x80x94CH2COxe2x80x94 group at the N-terminal position. In accordance with such an embodiment, Xaa1 is preferably Trp and Xaa2 is preferably Leu. Exemplary and preferred such peptides have the amino acid residue sequence of SEQ ID NO:9-12.
In another embodiment, a cyclic peptide of this invention further comprises two or more cysteine or modified cysteine residues and is cyclized via a disulfide bond. Preferably, one of the cysteine or modified cysteine residues is located at the N- or C-terminal position. In one embodiment, a cyclic peptide having two cysteine or modified cysteine residues comprises the amino acid residue sequence Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa1 and Xaa2 and Xaa3 are as defined above. Preferably, Xaa1 is Tyr, Phe, Trp, dTrp or Ile; Xaa2 is Leu, Ile, Val, Lys, Met or Asp; and Xaa3 is Val, Tyr, Leu, Trp, or Phe.
In yet another embodiment, a cyclic peptide having a cysteine or modified cysteine residue at both the N- and C-terminal positions comprises the amino acid residue sequence of SEQ ID NO:1. Preferably, Xaa1 is Trp or Phe, Xaa2 is Leu, Xaa3 is Val or Tyr and Xaa4 is Ser or Glu.
Exemplary and preferred cyclic, disulfide peptides of the present invention have the amino acid residue sequence of any of SEQ ID NO:13 or 15-43.
In another aspect, the present invention provides a pharmaceutical composition comprising a physiologically acceptable diluent and a cyclic peptide of the present invention. Preferred cyclic peptides in such a composition are the same as set forth above.
In yet another aspect, the present invention provides a process of selectively inhibiting the binding of xcex14xcex21 integrin to VCAM-1. That process comprises exposing a cell that expresses xcex14xcex21 integrin and a cell that expresses VCAM-1 to an effective inhibiting amount of a cyclic peptide of the present invention. In a preferred embodiment of that process, the VCAM-1 is on the surface of a vascular endothelial cell. In another preferred embodiment, the xcex14xcex21 integrin is on the surface of a white blood cell such as a monocyte, a lymphocyte, a granulocyte (an eosinophil or a basophil), a stem cell or other cell that naturally expresses xcex14xcex21. Preferred peptides used in that process are the same as set forth above.
Where the cells are located in a living organism, a peptide is preferably administered to the organism in an effective inhibiting amount in a pharmaceutical composition of this invention.
In another aspect, the present invention provides a process of selectively inhibiting the binding of xcex14xcex21 integrin to a protein comprising exposing the integrin to the protein in the presence of an effective inhibiting amount of a cyclic peptide of the present invention. Preferably, the xcex14xcex21 integrin is expressed on the surface of a cell such as a white blood cell or stem cell and the protein is part of the extracellular matrix such as fibronectin.
The present invention provides a process of inhibiting the binding of xcex14xcex21 integrin to proteins such as VCAM-1, fibronectin and invasin. The invention also provides cyclic peptides that inhibit that binding.
The adhesion of leukocytes to the vascular endothelium and their subsequent extravasation into tissues are critical steps in the inflammatory response. Vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin superfamily, is expressed by endothelial cells and a restricted number of other cell types. VCAM-1 can be induced by cytokines such as tumor necrosis factor-xcex11, interleukin-4, and interleukin-1xcex2 and is therefore hypothesized to contribute to leukocyte extravasion in inflammatory conditions such as rheumatoid arthritis, asthma, and atherosclerosis.
One molecular form of VCAM-1 contains seven immunoglobulin modules in its extracellular domain. VCAM-1 is recognized by the integrin receptor xcex14xcex21. xcex14xcex21 is expressed principally by leukocytes (T and B lymphocytes, monocytes, basophils, and eosinophils), and is also functional on mast cells, derivatives of the embryonic neural crest, and in developing muscle.
xcex14xcex21 also recognizes the extracellular matrix glycoprotein fibronectin. Three distinct xcex14xcex21-binding sites have been identified within fibronectin and all have been reproduced in synthetic form. One site (represented by the peptide H1) is found in the Hepil region and is therefore expressed in all fibronectin isoforms; two others (represented by peptides CS1 and CS5) are present in the alternatively spliced type III connecting segment. Of these three the CS1 peptide has the higher affinity for xcex14xcex21 and contains the tripeptide Leu-Asp-Val (LDV) as its minimal active site. H1 contains a related motif, IIe-Asp-Ala (IDA), while CS5 incorporates a variant of the prototypic RGD motif, Arg-Glu-Asp-Val.
In one aspect, the present invention provides cyclic peptides that inhibit binding of the xcex14xcex21 integrin to VCAM-1. A peptide of the present invention is modeled after the Leu-Asp-Val (LDV) domain of the CS1 peptide sequence, which domain is presented in such a way by the cyclic peptide to produce a potent xcex14xcex21 binding inhibitor.
Peptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino (N) to the carboxyl (C) terminus. Amino acid residue sequences are denominated by either a single letter or a three letter code. The meanings of those codes as well as various other abbreviations used herein are in accordance with the recommendation of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, and are shown below.
Modifications and changes can be made in the structure of a peptide of the present invention and still obtain a molecule that inhibits the binding of xcex14xcex21 integrin to VCAM-1. For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity; likewise, D- or L-amino acid residues can be used. D-amino acids are indicated herein as d-Xaa, where Xaa is the three-letter amino acid code (e.g., dTrp). In fact, certain amino acids can be substituted or added which greatly enhance binding inhibition. Because it is the interactive capacity and nature of a peptide that defines that peptide""s biological functional activity, certain amino acid sequence substitutions can be made in a peptide sequence and nevertheless obtain a peptide with like properties, particularly inhibition of the binding of xcex14xcex21 integrin to VCAM-1. Exemplary such peptides are set forth below.
A peptide contemplated by the present invention is cyclic. A cyclic peptide has a ring structure formed between certain amino acid residues of the corresponding linear peptide and can be envisioned as a linear peptide that is cyclized by covalent bonding of amino acid residues as described herein.
A cyclic peptide of the present invention contains from 4 to about 13 amino acid residues. The N-terminal amino acid residue has a free terminal amine group (NH2), acetyl group (Ac) or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight (MPEGX000) linked through an amide bond to the N-terminal residue. The C-terminal amino acid residue has a terminal carboxylic acid group (OH) or amide group. A cyclic peptide comprises the amino acid residue sequence Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa1 is any L- or D-xcex1-amino acid residue and Xaa2 and Xaa3 are any hydrophobic, L-xcex1-amino acid residue with the proviso that when Xaa1 is Lys or Arg, Xaa2 cannot be Gly or Cys. In a preferred embodiment, Xaa1 is Phe, Trp or Ile; Xaa2 is Leu, Ile, Val, Lys or Met; and Xaa3 is Val, Tyr, Leu, Trp or Phe. More preferably, Xaa1 is Trp, Xaa2 is Leu and Xaa3 is Val.
A peptide in accordance with the sequence set forth above can be extended in the N-terminal direction by the addition of from 1 to 5 L- or D-xcex1-amino acids and, in the C-terminal direction by the addition of from 1 to 5 L- or D-xcex1-amino acids.
A peptide can be cyclized without or with a sulfur containing bridge. Where a cyclic peptide does not comprise a sulfur containing bridge, the N- and C-terminal amino acid residues are joined together with an amide bond (formally a lactam in the case of cyclization). Where a cyclic peptide comprises a sulfur containing bridge, either one or two amino acid residues of the corresponding linear peptide is a Cys or modified Cys residue (dCys or dPen). Such a cyclic peptide can comprise a cyclic sulfide, sulfoxide or sulfone (one Cys residue in the corresponding linear peptide) or a cyclic disulfide (two Cys residues in the corresponding linear peptide).
Where cyclization has occurred by formation of a lactam by condensation of the N and C terminus, that peptide comprises the amino acid residue sequence:
Xaa4-Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:1),
where Xaa1 is any L- or D-xcex1-amino acid residue; Xaa2 and Xaa3 are independently any hydrophobic, L-xcex1-amino acid residue with the proviso that when Xaa1 is Arg or Lys, Xaa2 cannot be Gly or Cys; and Xaa4 is any D- or L-xcex1-amino acid. In a preferred embodiment, Xaa1 is Phe or Trp, Xaa2 is Leu, Xaa3 is Val and Xaa4 is Glu or Ser. Exemplary and preferred peptides in accordance with SEQ ID NO:1 have the sequences Glu-Trp-Leu-Asp-Val-Pro (SEQ ID NO:2), Glu-Trp-Leu-Asp-Val-Asp (SEQ ID NO:3), Glu-Trp-Leu-Asp-Val (SEQ ID NO:4), Glu-Trp-Leu-Asp-Asp (SEQ ID NO:5), Glu-Trp-Leu-Asp-Val-Pro-Glu-Trp-Leu-Asp-Val (SEQ ID NO:6), Gly-Pro-Glu-Phe-Leu-Asp-Val (SEQ ID NO:7) and Glu-Phe-Leu-Asp-Val (SEQ ID NO:8).
Where a peptide of the present invention contains a sulfide, sulfoxide or sulfone bridge, that peptide comprises a cysteine or modified cysteine residue at one position and a xe2x80x94CH2COxe2x80x94 group at the N-terminal position. As used herein, the term xe2x80x9cmodified cysteinexe2x80x9d refers to D-cysteine (dCys) or D-penicillamine (dPen). The sulfur atom of the cysteine or modified cysteine residue is attached to the CH2 group forming the cyclic peptide.
Such a peptide comprises the amino acid residue sequence Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa1, Xaa2 and Xaa3 are as defined above. Preferably, Xaa1 is Trp, Xaa2 is Leu and Xaa3 is Val or Cys. Exemplary and preferred peptides have the sequences CH2CO-Ser-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:9), CH2CO-Glu-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:10), CH2CO-Glu-trp-Leu-Asp-Cys-acid (SEQ ID NO:11), and CH2CO-Trp-Leu-Asp-Val-Cys-COOH (SEQ ID NO:12).
Where a peptide of the present invention contains a disulfide bridge, that peptide contains two cysteine or modified cysteine residues. Preferably, one of the cysteine or modified cysteine residues is located at the N- or C-terminal position and at least one of those amino acid residues is Cys. Such a disulfide peptide comprises the amino acid residue sequence Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa1, Xaa2 and Xaa3 are as defined above. Preferably, Xaa1 is Trp or Cys, Xaa2 is Leu and Xaa3 is Val or Cys. Exemplary such peptides have the sequence Cys-Leu-Asp-Val-Cys (SEQ ID NO:13) or Cys-Trp-Leu-Asp-Cys-acid (SEQ ID NO:14).
In another preferred embodiment of SEQ ID NO:15, Xaa1 is Tyr, Phe, Trp, dTrp or Ile; Xaa2 is Leu, Ile, Val, Lys, Met or Asp; and Xaa3 is Val, Tyr, Leu, Trp, or Phe. More preferably, a disulfide cyclic peptide has the sequence Cys-Ser-Trp-Leu-Asp-Val-Cys (SEQ ID NO:16), Cys-dTrp-Leu-Asp-Val-Cys-acid (SEQ ID NO:17), Ac-Cys-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:18), Cys-Tyr-Leu-Asp-Val-Cys-acid (SEQ ID NO:19), Cys-Trp-Leu-Asp-Phe-Cys-acid (SEQ ID NO:20), Cys-Phe-Leu-Asp-Val-Cys-acid (SEQ ID NO:21), Cys-Trp-Leu-Asp-Trp-Cys-acid (SEQ ID NO:22), Cys-Trp-Ile-Asp-Val-Cys-acid (SEQ ID NO:23), Cys-Trp-Met-Asp-Val-Cys-acid (SEQ ID NO:24), Cys-Trp-Val-Asp-Val-Cys-acid (SEQ ID NO:25), Cys-Trp-Lys-Asp-Val-Cys-acid (SEQ ID NO:26), Cys-Trp-Leu-Glu-Val-Cys-acid (SEQ ID NO:27), Cys-Trp-Leu-Asp-Leu-Cys-acid (SEQ ID NO:28), Cys-Trp-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:29), Cys-Ile-Leu-Asp-Val-Cys-acid (SEQ ID NO:30), Cys-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:31), and dCys-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:32).
In another preferred embodiment, a disulfide cyclic peptide of the present invention comprises the amino acid residue sequence of SEQ ID NO:1, above. Preferably, Xaa1 is Trp or Phe, Xaa2 is Leu, Xaa3 is Val or Tyr and Xaa4 is Ser or Glu. Exemplary and preferred such peptides have the sequence of Cys-Glu-Trp-Leu-Asp-Val-Cys-amide (SEQ ID NO:33), Cys-Glu-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:34), dCys-Glu-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:35), Cys-Glu-Trp-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:36), Cys-Ser-Phe-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:37), Cys-Glu-Phe-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:38), dCys-Ser-Trp-Leu-Asp-Val-dCys-acid (SEQ ID NO:39); Cys-Pro-Glu-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:40), MPEG5000-Cys-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:41) and dPen-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:42).
In another embodiment, a cyclic disulfide peptide of the present invention comprises the sequence:
Xaan-Cys-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:43),
where Xaa is any D- or L-xcex1-amino acid and n is an integer from 1 to 7.
A peptide of the present invention can be made using standard peptide synthetic procedures well known in the art. Typically, peptides were made with Fmoc-amino acids. However, peptides can also be made using Boc protecting groups by methods well known to those skilled in the art. Side chain protecting groups of trifunction amino acids used in the synthetic procedure include Arginine (Pmc), Aspartic acid (tBu), Cysteine (Trt), Glutamic acid (tBu), Histidine (Boc), Lysine (Boc), Serine (tBu), Threonine (tBu), and Tyrosine (tBu). Other protecting groups are specifically described.
The preparation of the peptides in this invention by solid phase methodology is well known to those skilled in the art, and can be described as follows. Peptides were synthesized on an insoluble carrier such as p-benzyloxybenzyl alcohol resin for the synthesis of C-terminal carboxylic acid peptides (Wang resin, where normally the resin can be purchased with the first amino acid bound), and 4-(2xe2x80x2,4xe2x80x2-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin for C-terminal amide peptides (Rink resin). The peptides were prepared by solid phase synthesis using either HBTU or DIC chemistry procedures on a Protein Technologies Inc. Symphony peptide synthesizer.
The C-terminal amide peptides were prepared by coupling the C-terminal amino acid of the sequence to the Rink resin using the same general method as the other couplings. The C-terminal carboxylic acid peptides were prepared by purchasing Wang resin to which the C-terminal amino acid was bound to the resin as a carboxylic ester. The xcex1-amino protecting group was removed by piperidine treatment, and the next Fmoc-amino acid coupled to the resin by simultaneous treatment of the resin with the Fmoc-amino acid, a coupling reagent such as DIC or HBTU, and if necessary HOBT. Such deprotection and couplings were repeated to afford each desired peptide. In all cases the Fmoc protecting group was removed by treatment with a 20% solution of piperidine in DMF. However, it is understood by those skilled in the art that the exact percentage of piperidine is not critical and should not be considered limiting in this invention. It is also understood by those skilled in the art that piperidine can be replaced by other bases, furthermore the coupling reagents and protocols used can be substituted with any of those known in the field of peptide synthesis (including the use of Boc chemistry based solid phase synthesis and also solution phase peptide synthesis), and those reagents specifically used in the examples provided should not be considered limiting for this invention. All unnatural amino acids, D-amino acids and other compounds were coupled by manual addition of the reagent, following the same procedure as for automated operation.
Where cyclic sulfides, sulfoxides, sulfones or disulfides with C-terminal carboxylic acids are desired, the peptides can be synthesized on an insoluble carrier such as p-benzyloxybenzyl alcohol resin (Wang resin), whereas the equivalent C-terminal amides were prepared on 4-(2xe2x80x2,4xe2x80x2-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink resin). Where two cysteines are present, mild acid removal of the trityl protecting groups and oxidative cyclization on the resin using DMSO or NIS forms the disulfide bond, and this compound was cleaved from the resin in the normal way. Alternatively disulfides can be prepared by solution phase cyclization of the linear sequence in guanidine hydrochloride. Where cyclic sulfides (and their oxidation products) are desired the N-terminus can be acylated with bromoacetic acid, the cysteine trityl group removed and cyclization achieved by NMM in DMF treatment. The head-tail lactams were synthesized on a chlorotrityl resin which forms a carboxylic ester linkage between the C-terminal amino acid and the resin. The linear peptide was cleaved from the resin with acetic acid in DCM and cyclized in solution to form the lactam.
Peptides were cleaved from the resin with a TFA cocktail after the removal of the N-terminal Fmoc protecting group. The exact composition of the TFA cocktail was varied depending on the side chain protecting groups present, and is well known to those skilled in the art. The range of TFA was from 85 to 95%, and the remainder comprised of a mixture of scavengers selected from a combination of anisole, thioanisole, cresol, thiocresol, phenol, thiophenol, EDT, trimethylsilane and water. The time of the cleavage reaction required is sequence dependant, normally being from 1 to 3 hours. After cleavage, the resin was removed by filtration and cold ether added to the solution to give a precipitate. The precipitate was collected and washed a few times with ether to remove residual TFA and scavengers. The precipitate was redissolved in aqueous solution for lyophilization to give the crude product.
Purification was carried out by reverse-phase HPLC on a C18-preparative column (300 xc3x85, 21.4 mmxc3x9725 cm, 5 xcexcm spherical packing) at a flow rate of 10 ml/min. The selection of any other suitable packing known to one skilled in the art is equally acceptable. Products were detected by UV absorption at 214 nm. Two mobile phases were used in the HPLC system, solution A and B using a gradient elution. Solution A was comprised of 5% acetonitrile in deionized water containing 0.15% TFA, while solution B was comprised of 5% deionized water in acetonitrile containing 0.1% of TFA. A gradient of increasing percentage of solution B was used to elute the peptide from the solid support, however the gradient used is sequence dependant. Other methods of purification known to one skilled in the art are equally acceptable. The purity of the peptide was checked by C18 analytical HPLC (300 xc3x85, 4.6 mmxc3x9725 cm, 5 xcexcm spherical packing) at a flow rate of 1 ml/min.
A detailed description of the synthesis of exemplary peptides is set forth hereinafter in the Examples.
In another aspect, the present invention provides a pharmaceutical composition comprising a peptide of the present invention and a physiologically tolerable diluent.
The present invention includes one or more peptides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for intranasal delivery, for oral administration in solid or liquid form, for rectal or topical administration, or the like.
The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol.
The compositions can also be delivered through a catheter for local delivery at a target site, via an intracoronary stent (a tubular device composed of a fine wire mesh), or via a biodegradable polymer. The compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions.
The compositions are preferably administered by catheter, i.v. or subcutaneous injection, or intranasally via a spray or aerosol.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents.
Suspensions, in addition to the active compounds, can contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
In another aspect, the present invention contemplates a process of selectively inhibiting the binding of xcex14xcex21 integrin to VCAM-1. A process of the present invention can be used in vitro or in vivo in a living organism. In accordance with a process of the present invention, a cell expressing xcex14xcex21 integrin is exposed to a cell expressing VCAM-1 in the presence of an effective inhibiting amount of a peptide of the present invention. Means for determining an effective inhibiting amount are well known in the art.
A cell expressing xcex14xcex21 integrin can be a naturally occurring white blood cell, mast cell or other cell type that naturally expresses xcex14xcex21 on the cell surface, or a cell transfected with an expression vector that contains a polynucleotide (e.g., genomic DNA or cDNA) that encodes xcex14xcex21 integrin. In an especially preferred embodiment, xcex14xcex21 integrin is present on the surface of a white blood cell such as a monocyte, a lymphocyte or a granulocyte (e.g., an eosinophil or a basophil).
A cell that expresses VCAM-1 can be a naturally occurring cell (e.g. an endothelial cell) or a cell transfected with an expression vector containing a polynucleotide that encodes VCAM-1. Methods for producing transfected cells that express VCAM-1 are well known in the art.
Where VCAM-1 exists on the surface of cell, the expression of that VCAM-1 is preferably induced by inflammatory cytokines such as tumor necrosis factor-xcex1, interleukin-4 and interleukin-1xcex2.
Where the cells expressing xcex14xcex21 integrin and VCAM-1 are in a living organism, a peptide is administered in an effective amount to the living organism. Preferably, the peptide is in a pharmaceutical composition of this invention. Administering is preferably accomplished via intravascular injection or intranasal administration.
A process of the present invention is especially useful in treating diseases associated with uncontrolled migration of white blood cells to damaged tissue. Such diseases include, but are not limited to, asthma, atherosclerosis, rheumatoid arthritis, allergy, multiple sclerosis, leukemia, and brain cancer.
A process of inhibiting VCAM-1 and xcex14xcex21 binding uses a cyclic peptide of the present invention as set forth hereinbefore. Preferred such peptides are the same as set forth above. More preferably, a peptide used in a process of the present invention has the amino acid residue sequence of SEQ ID NO:2-14 or 16-42. Even more preferably, peptides have the amino acid residue sequence of SEQ ID NO:3-7, 9-12, 16-26, 28, 29 or 31-42.
The present invention also provides a process of selectively inhibiting the binding of xcex14xcex21 integrin to a protein comprising exposing the integrin to the protein in the presence of an effective inhibiting amount of a cyclic peptide of the present invention. In a preferred embodiment, the xcex14xcex21 integrin is expressed on the surface of a cell, either naturally occurring or a cell transformed to express xcex14xcex21 integrin.
The protein to which the xcex14xcex21 integrin binds can be expressed on a cell surface or part of the extracellular matrix. Especially preferred proteins are fibronectin or invasin. Preferred peptides for use in such a process are the same as set forth above.
The ability of peptides of the present invention to inhibit binding are described in detail hereinafter in the Examples.